Investigating the role of the Ras/MAPK pathway in increasing immunogenicity of triple negative breast cancers

Abstract

The predictive and prognostic significance of tumour infiltrating lymphocytes (TILs) has been highlighted in various solid cancers in recent years, where TIL infiltration has become a landmark biomarker of survival outcomes and response to therapy. These findings suggest an important role for T cell mediated immunosurveillance in influencing the biology of these cancers. More recently, studies have also demonstrated the prognostic value of TILs in certain breast cancer subtypes such as HER2-positive cancers, and in particular, triple negative breast cancer (TNBC), where the presence of higher levels of TILs in primary tumours was found to correlate with better disease free and overall survival. These associations suggest that immunotherapies may be effective in TNBC, a breast cancer subtype where novel therapies are urgently needed.

Studies from our group have shown that oncogenic activation of the Ras/MAPK pathway is associated with significantly decreased levels of TILs and poorer survival in TNBC patients. This observation raises the possibility that Ras/MAPK pathway inhibition may relieve local immunosuppression, thereby enhancing TIL infiltrate and improving patient outcomes. Several studies undertaken through this thesis, in human and mouse TNBC cell lines, revealed that MEK inhibition (MEKi) was efficient at increasing tumour antigenicity, where small disruptions to MAPK signaling were able to enhance expression of immune markers such as MHC-I. Paradoxically, MEK signaling in lymphocytes is critical for CD8+ and CD4+ T cell activation, proliferation, function and survival. Therefore, the studies in this thesis aimed to investigate the effect of MEKi on tumour antigenicity, as well as the long-term effects of MEKi on T cell function. Although MEKi increased tumour antigenicity, interestingly, the data demonstrated that MEKi elicited inhibitory effects in both mouse and human T cells.

It was hypothesised that agonist antibodies such as α-4-1BB (CD137) and α-OX-40 (CD134) may rescue T cell function in the presence of MEKi. The studies undertaken in Chapter 3 definitively demonstrated that MEKi significantly inhibits early T cell signaling, where the administration of immune agonists α-4-1BB and α-OX-40 in combination with MEKi, effectively maintained T cell frequency, proliferation and function after TCR stimulation, through the activation of alternative signaling pathways. As such, these results confirm that MEKi can prime tumour immunogenicity, and combination with either α-4-1BB or α-OX-40 agonist immunotherapy results in superior therapeutic efficacy due to protection of early and crucial TIL function in preclinical models of TNBC.

Given the inhibitory effect of MEKi on T cells, the work in Chapter 4 endeavoured to develop a model where the effect of MEK1/2 knockdown (MEK KD) could be achieved intrinsically within the tumour, without affecting normal T cell function. This model would therefore allow the investigation into the genes that underpin the relationship between the Ras/MAPK pathway and tumour cell immunogenicity. Using this model, several antigen processing and presentation pathway genes were found to be increased following MEKi and MEK KD in tumours. Notably, immunoproteasome components were found to be upregulated following MEKi or MEK KD, suggesting that MEK suppression promotes the conversion of the proteasome to an immunoproteasome, thereby increasing the number of MHC-I related peptides for presentation to CD8+ T cells. Furthermore, RNAseq analysis of tumours cells with MEKi or MEK KD revealed that several extracellular matrix (ECM) remodelling genes and key chemokines were upregulated following MEK1/2 suppression.

In summary, this work has characterised the ability of MEKi to enhance antigenicity of TNBCs, as well as deciphering mechanisms behind the detrimental effects involved with MEKi on T cell functional responses. Moreover, the studies conducted within this thesis highlight the strategies that may be used in order to overcome this inhibitory effect, as well as the alternative MAPK pathways involved in T cell rescue with agonist antibodies; through p38 and JNK signaling. Additionally, important factors involved in antigen processing and presentation and immunoproteasome conversion, associated with MEKi were discovered. This potentially explains the clinical observation that RAS/MAPK activation is associated with poor TIL infiltrate and suggests that novel targets could be validated, and future drugs developed to enhance antigen presentation in cancer, that could salvage or bypass the global effects of MEKi on T cells. Moreover, the principle of exploiting ECM stiffness and degradation to permit greater TIL infiltration and function, or enhanced drug penetration, may be further developed as a potential therapeutic strategy for future treatment approaches in TNBC, particularly those which harbour mutations that activate the RAS/MAPK pathway.